The polymerase chain reaction (PCR) is an in vitro method for producing large amounts of a specific DNA fragment of defined length and sequence from small amounts of a complex template. Recombinant DNA techniques have revolutionized molecular biology and genetics by permitting the isolation and characterization of specific DNA fragments. Many cloning methods can be complemented and sometimes even circumvented by using PCR, and novel applications of the technique now permit studies that were not possible before. Such methods include DNA fragment isolation, fragment endlabeling, mutagenesis, DNase I footprinting, cDNA cloning, genomic cloning, promoter manipulations, DNA sequencing, and RNA and DNA quantitation. The sensitivity, speed and versatility of PCR are having a profound impact on molecular biological approaches to problems in human genetics, forensic science, and evolutionary and developmental biology.
PCR is based on the enzymatic amplification of a DNA fragment that is flanked by two oligonucleotide primers that hybridize to opposite strands of the target sequence. The primers are oriented with their 3' ends pointing towards each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences and extension of the annealed primers with a DNA polymerase result in the amplification of the segment defined by the 5' ends of the PCR primers. Since the extension product of each primer can serve as a template for the other primer, each cycle essentially doubles the amount of the DNA fragment produced in the previous cycle. This results in the exponential accumulation of the specific target fragment, up to several millionfold in a few hours. The method can be used with a complex template such as genomic DNA and can amplify a single-copy gene contained therein. It is also capable of amplifying a single molecule of target in a complex mixture of RNAs or DNAs and can, under some conditions, produce fragments up to ten kbp long. The PCR technology is the subject matter of U.S. Pat. Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202 all of which are incorporated by reference herein.
By using the thermostable Taq DNA polymerase isolated from the thermophilic bacterium Thermus aquaticus instead of the E. coli Klenow fragment of DNA polymerase I, it has been possible to avoid inactivation of the polymerase which necessitated the addition of enzyme after each heat denaturation step. This development has led to the automation of PCR by a variety of simple temperature-cycling devices, and consequently the use of PCR has expanded rapidly. In addition, the specificity of the amplification reaction is increased by allowing the use of higher temperatures for primer annealing and extension. The increased specificity improves the overall yield of amplified products by minimizing the competition by nontarget fragments for enzyme and primers.
While the possible uses of PCR are numerous, the applications are limited to those situations where enough is known about the DNA sequence to design two PCR primers which hybridize to opposite strands of the target sequence. Thus, in techniques such as sequencing, footprinting, or cloning promotor elements, where one end of the template is unknown PCR cannot be utilized.
Thus, there exists a need for an effective method to add a uniform, defined sequence which would allow for the use of PCR when only one end sequence is initially known. Such a method could be of critical importance to increasing the efficiency of sequencing the human genome. The present invention satisfies this need and provides related advantages as well.